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Global Reprogramming of Gene Transcription in Trichoderma reesei by Overexpressing an Artificial Transcription Factor for Improved Cellulase Production and Identification of Ypr1 as an Associated Regulator.

ABSTRACT: Synthetic biology studies on filamentous fungi are providing unprecedented opportunities for optimizing this important category of microbial cell factory. Artificial transcription factor can be designed and used to offer novel modes of regulation on gene transcription network. Trichoderma reesei is commonly used for cellulase production. In our previous studies, a plasmid library harboring genes encoding artificial zinc finger proteins (AZFPs) was constructed for engineering T. reesei, and the mutant strains with improved cellulase production were selected. However, the underlying mechanism by which AZFP function remain unclear. In this study, a T. reesei Rut-C30 mutant strain T. reesei U5 bearing an AZFP named as AZFP-U5 was focused, which secretes high level protein and shows significantly improved cellulase and xylanase production comparing with its parental strain. In addition, enhanced sugar release was achieved from lignocellulosic biomass using the crude cellulase from T. reesei U5. Comparative transcriptome analysis was further performed, which showed reprogramming of global gene transcription and elevated transcription of genes encoding glycoside hydrolases by overexpressing AZFP-U5. Furthermore, 15 candidate regulatory genes which showed remarkable higher transcription levels by AZFP-U5 insertion were overexpressed in T. reesei Rut-C30 to examine their effects on cellulase biosynthesis. Among these genes, TrC30_93861 (ypr1) and TrC30_74374 showed stimulating effects on filter paper activity (FPase), but deletion of these two genes did not affect cellulase activity. In addition, increased yellow pigment production in T. reesei Rut-C30 by overexpression of gene ypr1 was observed, and changes of cellulase gene transcription were revealed in the ypr1 deletion mutant, suggesting possible interaction between pigment production and cellulase gene transcription. The results in this study revealed novel aspects in regulation of cellulase gene expression by the artificial regulators. In addition, the candidate genes and processes identified in the transcriptome data can be further explored for synthetic biology design and metabolic engineering of T. reesei to enhance cellulase production.

SUBMITTER: Zhang F

PROVIDER: S-EPMC7351519 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Global Reprogramming of Gene Transcription in Trichoderma reesei by Overexpressing an Artificial Transcription Factor for Improved Cellulase Production and Identification of Ypr1 as an Associated Regulator.

Zhang Fei F Li Jia-Xiang JX Champreda Verawat V Liu Chen-Guang CG Bai Feng-Wu FW Zhao Xin-Qing XQ

Frontiers in bioengineering and biotechnology 20200703

Synthetic biology studies on filamentous fungi are providing unprecedented opportunities for optimizing this important category of microbial cell factory. Artificial transcription factor can be designed and used to offer novel modes of regulation on gene transcription network. Trichoderma reesei is commonly used for cellulase production. In our previous studies, a plasmid library harboring genes encoding artificial zinc finger proteins (AZFPs) was constructed for engineering T. reesei< ...[more]

PMID: 32719779

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Project description:BackgroundTrichoderma reesei is widely used for cellulase production and accepted as an example for cellulase research. Cre1-mediated carbon catabolite repression (CCR) can significantly inhibit the transcription of cellulase genes during cellulase fermentation in T. reesei. Early efforts have been undertaken to modify Cre1 for the release of CCR; however, this approach leads to arrested hyphal growth and decreased biomass accumulation, which negatively affects cellulase production.ResultsIn this study, novel fusion transcription factors (fTFs) were designed to release or attenuate CCR inhibition in cellulase transcription, while Cre1 was left intact to maintain normal hyphal growth. Four designed fTFs were introduced into the T. reesei genome, which generated several transformants, named Kuace3, Kuclr2, Kuace2, and Kuxyr1. No obvious differences in growth were observed between the parent and transformant strains. However, the transcription levels of cel7a, a major cellulase gene, were significantly elevated in all the transformants, particularly in Kuace2 and Kuxyr1, when grown on lactose as a carbon source. This suggested that CCR inhibition was released or attenuated in the transformant strains. The growth of Kuace2 and Kuxyr1 was approximately equivalent to that of the parent strain in fed-batch fermentation process. However, we observed a 3.2- and 2.1-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of the parent strain. Moreover, we observed a 6.1- and 3.9-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of Δcre1 strain.ConclusionsA new strategy based on fTFs was successfully established in T. reesei to improve cellulase titers without impairing fungal growth. This study will be valuable for lignocellulosic biorefining and for guiding the development of engineering strategies for producing other important biochemical compounds in fungal species.

Project description:BackgroundTrichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging.ResultsComparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1.ConclusionsThe regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Dataset Information

Global Reprogramming of Gene Transcription in Trichoderma reesei by Overexpressing an Artificial Transcription Factor for Improved Cellulase Production and Identification of Ypr1 as an Associated Regulator.

Publications

Global Reprogramming of Gene Transcription in <i>Trichoderma reesei</i> by Overexpressing an Artificial Transcription Factor for Improved Cellulase Production and Identification of Ypr1 as an Associated Regulator.

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